Transcriptomic Profiling Reveals the Impact of ASIC2 Knockdown in Primary Mouse Thalamic Neurons

This study investigates the transcriptomic consequences of knocking down the Acid-Sensing Ion Channel Subunit 2 (ASIC2) gene in primary mouse thalamic neurons. Primary thalamic neurons were isolated and cultured at a density of 500,000 cells per well. Three days post-plating, ASIC2 expression was knocked down using specific shRNA constructs. Following the knockdown period, total RNA was extracted from the cells and subjected to bulk mRNA sequencing (RNA-seq) to comprehensively analyze changes in global gene expression profiles. This experimental design aims to identify differentially expressed genes and pathways regulated by ASIC2 in thalamic neurons, providing insights into its functional roles within this specific neuronal population. Primary thalamic neurons were isolated from mice (e.g., postnatal C57BL/6 mice, P0-P2), dissociated, and plated at a density of 500,000 viable cells per well onto poly-D-lysine/laminin-coated culture plates (e.g., 24-well plates). The neurons were cultured in Neurobasal-based medium supplemented with B27, GlutaMAX, and penicillin/streptomycin, and maintained at 37°C with 5% CO₂. Three days after plating (DIV 3), the neurons were transduced or transfected (e.g., via lentiviral transduction or lipid-based transfection) with ASIC2-specific shRNA or siRNA constructs (experimental group) or non-targeting/scrambled sequence constructs (control group). Following transfection, the cells were cultured for an additional 48-72 hours (e.g., until DIV 5 or DIV 6) to ensure efficient gene knockdown. Subsequently, cells were harvested, gently washed with PBS, and lysed directly in the wells using an RNA lysis buffer (e.g., TRIzol).